This proposal focuses on endocytic processes that remove transporters, specifically cardiac Na/Ca exchangers (NCX1), from the surface membrane. Membrane fusion and budding processes are fundamental to all eukaryotic life, and we have developed improved electrophysiological methods to analyze trafficking events at the cell surface, starting with immortalized fibroblasts and proceeding to adult cardiac myocytes. Exploiting unprecedented control of the cytoplasmic milieu with high resolution capacitance recording, we have discovered that cytoplasmic ATP depletion, followed by a Ca transient and ATP replenishment, promotes a massive endocytic response (MEND). We have further determined that NCX1 is internalized during MEND. As NCX1 plays a major role in ischemia-reperfusion damage and related cardiac arrhythmias, removal of NCX1 from the membrane in response to metabolic stress can be of substantial clinical significance. Therefore, we have initiated a detailed analysis of the MEND response. Preliminary Data indicates that MEND is driven by remodeling of actin membrane cytoskeleton with ATP-, Ca- and PIP2- dependent processes all playing essential roles. Further Preliminary Data shows that NCX1 lateral mobility decreases dramatically in steps leading up to MEND, as well as with stabilization of F-actin. Therefore, we will analyze how metabolic state regulates actin cytoskeleton and NCX1-actin cytoskeleton interactions. Additionally, we will identify the Ca sensors underlying MEND, and we will analyze how PIP-kinases involved in MEND are regulated. To address how NCX1 couples to MEND, new NCX1 fusion proteins have been developed for on-line monitoring of NCX1 internalization, pulse-chase tracking of NCX1, and improved analysis of NCX1 mobility. An NCX1 fusion with Dendra2 allows conversion of green transporters to red transporters, followed by tracking of the two transporter species. Halotag fusions on the extracellular side allow sequential NCX1 labeling with different membrane-permeable and -impermeable fluorophores. In the longer term, these fusions will allow the use of quantum dots and Nanogold to study NCX1 trafficking. Overall, the proposed work will generate fundamental insights into a powerful endocytic process that is of wide cell biological interest and is likely to play an important role in cardiac ischemia-reperfusion and related pathologies.